Expression and function of glial cell line derived neurotrophic factor family receptor alpha 1 alternatively spliced isoforms

EXPRESSION AND FUNCTION OF GLIAL CELL-LINE DERIVED NEUROTROPHIC FACTOR FAMILY RECEPTOR ALPHA ALTERNATIVELY SPLICED ISOFORMS PENG ZHONG NI (B Sc., UWA) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2003 Acknowledgements I would like to express my greatest gratitude to my supervisor, Associate Professor Too Heng-Phon for giving me this opportunity to pursue my interest in science and further study His guidance and supports for my research project have been tremendous and invaluable I would like to thank specially the lab members, Jason, Thai, Nivetha and Lee Foong for their cooperation, assistance and encouragement I appreciate the great help from the staff and friends of the department, and the inspiring, encouraging and friendly environment, which makes my stay memorable and enjoyable At last, I would like to express my deepest appreciation to my family, my dear husband Jun, my two lovely sons, Tony and Simon for their understanding, inspiring, patience and constant supporting Without them, this project would not be fulfilled i Contents Acnowledgements i Contents ii Summary vii List of tables ix List of figures ix Abbreviations xi Chapter 1: Introduction 1.1 Introduction 1.2 GDNF in Parkinson’s disease (PD) 1.3 GDNF family 1.3.1 GDNF family ligands (GFLs) 1.3.2 GDNF family receptors 1.3.3 c-Ret as a co-receptor for GDNF family 1.3.4 GDNF family multi-component receptor complex signaling 1.4 GDNF 1.4.1 Discovery and identification of GDNF 1.4.2 Neurotrophic effects of GDNF 1.5 GFRα1 9 10 11 1.5.1 GFRα1 and its spliced isoforms 11 1.5.2 Expression and functional role of GFRα1a 12 ii 1.6 Alternatively spliced isoforms 13 1.7 Quantification of gene expression 14 1.7.1 Quantification of gene expression at the transcription level 14 1.7.2 Absolute quantification using real time PCR 15 1.8 Aim of this study 16 Chapter 2: Materials and Methods 18 2.1 Molecular techniques 19 2.1.1 Agarose gel electrophoresis of DNA 19 2.1.2 DNA recovery from agarose gel 19 2.1.3 Ligation of DNA fragment with vector 20 2.1.4 Preparation of competent cells using calcium chloride 20 2.1.5 DNA transformation of E.coli cells by heat shock 21 2.1.6 Selection of recombinant DNA 21 2.1.7 Isolation of plasmid DNA 22 2.1.7.1 Alkaline lysis method (small scale preparation) 22 2.1.7.2 WizardTM Plus minipreps DNA purification system 22 2.1.7.3 WizardTM Plus midipreps DNA purification system 23 2.1.7.4 NucleoBond® Plasmids purification system 24 2.1.8 Restriction enzyme digestion of plasmid DNA 24 2.1.9 DNA sequencing 25 2.1.10 Total RNA extraction from mammalian cells using Quantum Prep AquaPureTM RNA isolation system 26 2.1.11 Agarose gel electrophoresis of RNA 26 2.1.12 Reverse transcription (RT) of RNA 27 iii 2.1.13 Polymerase chain reaction (PCR) amplification 2.2 Protein chemistry techniques 27 28 2.2.1 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) 28 2.2.2 Western blotting and detection 28 2.2.3 Protein quantification by BCA assay 29 2.3 Mammalian cell culture 30 2.3.1 Mammalian cell line 30 2.3.2 Cell transfection by FuGENE 6® Transfection Reagent 31 2.3.3 Haemocytometer for cell count 31 2.3.4 Cell Proliferation measured by Cell Proliferation BiotrakTM ELISA System 32 Chapter 3: Quantitation of GFRα1 Alternatively Spliced Isoforms, GFRα1a and GFRα1b by Real Time PCR 34 3.1 Introduction 35 3.2 Materials and Methods 37 3.2.1 Real time PCR 37 3.2.2 Standards for real time PCR 37 3.2.3 Reverse transcription (RT) of total RNA of human tissues and cell samples 38 3.2.4 Sequence dependent real time PCR using specific TaqMan probe for detection 38 3.2.5 Sequence independent real time PCR using SYBR Green I for detection 39 3.2.6 Statistical analysis 43 3.3 Results 3.3.1 Sequence dependent real time PCR quantitation using specific probes 3.3.1.1 Amplification efficiency using the common primers 43 43 44 iv 3.3.1.2 Determining the optimum concentration of the specific MGB TaqMan probe used for the detection 45 3.3.1.3 Detection with the specific MGB probe using common primers for amplification is not reliable for the two isoforms 3.3.2 Sequence independent real time PCR quantitation using SYBR Green I 45 47 3.3.2.1 Amplification specificity and efficiency of sequence independent real time PCR 47 3.3.2.2 Expression levels of GFRα1a, GFRα1b and c-Ret in various human tissues 51 Chapter 4: In Vitro Functional Study of The Two GFRα1 Spliced Receptor Isoforms 54 4.1 Introduction 55 4.2 Materials and Methods 56 4.2.1 Stably transfected cell lines of the two GFRα1 spliced isoforms 56 4.2.2 Quantitation of expression levels of the two GFRα1 spliced isoforms and c-Ret in the transfected cells by real time PCR 57 4.2.3 Morphological changes during differentiation of the transfected cell lines 57 4.2.4 Cell proliferation studies 58 4.2.5 Signaling pathway mapping 58 4.2.5.1 Cell stimulation by GDNF and NTN 58 4.2.5.2 Western blot analysis 59 4.3 Results 60 4.3.1 Expression levels of GFRα1a, GFRα1b and c-Ret in transfected Neuro2a cells 60 4.3.2 GDNF and NTN induced the differentiation of GFRα1a but not GFRα1b transfected cells 60 v 4.3.3 Cell proliferation profiles of GFRα1a and GFRα1b transfected cells were similar 64 4.3.4 Kinetic differences in the activation of Erk1/Erk2 signaling pathway between the two spliced isoforms 67 4.3.4.1 Dose response study 67 4.3.4.2 Time course study 68 Chapter 5: Discussion and Future work 70 References 76 Appendix I: Media and Buffers 84 Appendix II: Bacteria strain and Mammalian cell line 87 Appendix III: Gene Organization and Sequences 89 vi Summary Glial cell-line derived neurotrophic factor (GDNF) is a potent neurotrophic factor which shows restorative effects in a wide variety of rodent and primate models of Parkinson’s disease (PD) It promotes the survival of a broad range of central and peripheral neurons and is essential for kidney and enteric nervous system development, as well as regulating the fate of stem cells during spermatogenesis GDNF binds preferentially to GDNF family receptor alpha (GFRα1), which mediates the activation of the proto-oncogene c-Ret receptor protein-tyrosine kinase to form a multi-component system and trigger off downstream signaling events Two alternatively spliced GFRα1 isoforms, GFRα1a and GFRα1b have been previously identified GFRα1a and GFRα1b are highly homologous, with GFRα1a containing an extra 15 nucleotide (exon 5) compared to GFRα1b Currently, the specific physiological functional roles of GFRα1a and GFRα1b are unknown Further more, the distribution and expression levels of these two isoforms have not been reported To understand the physiological and functional role of GFRα1a and GFRα1b, stably transfected cell lines containing endogenous c-Ret were established to generate cells overexpressing either GFRα1a or GFRα1b Using the cell lines, mechanisms involved in the signaling pathways and functional roles of GFRα1a and GFRα1b in cell morphological differentiation and proliferation were investigated An isoform specific quantitative realtime PCR was used to confirm the existence and to measure the transcriptional expression levels of GFRα1a and GFRα1b in various human tissues vii The signaling pathway studies showed that cells expressing GFRα1a or GFRα1b when stimulated by specific GDNF family ligands (GFL), GDNF and Neurturin (NTN), resulted in rapid activation of MAPK (Erk1/2) and significantly different morphological changes A modurate but significant difference in the kinetics of the phosphorylation of Erk1/2 was detected when GFRα1a and GFRα1b transfected cells were exposed to NTN No significant changes in the proliferation profile in the transfected cells were observed compared to parental cells containing the vector only Real time PCR revealed that both GFRα1a and GFRα1b isoforms existed in all the human tissues examined, and the transcriptional expression levels of the two isoforms were similar in human fetal and adult brain GFRα1b levels were found to be much higher than GFRα1a in peripheral tissues In conclusion, the two spliced isoforms, GFRα1a and GFRα1b are differentially expressed in the human tissues examined A close investigation reveals that the both isoforms, despite having only amino acid sequence difference, show remarkable differences in their signaling and capabilities in inducing morphological differentiation viii List of Tables Table 3.1 Sequences and concentrations of the primers used in real time PCR reactions 42 List of Figures Fig.1.1 GDNF family ligands and their preferential receptors signal through Ret receptor tyrosine kinase Fig.1.2 NCAM as an alternative receptor for GDNF signaling receptor complex in cells lacking c-Ret Fig.3.1 Exon organization of human GFRα1a and GFRα1b spliced isoforms and locations of the primers and the specific MGB TaqMan probe 41 Fig.3.2 Exon organization of c-Ret isoforms, Ret 51, Ret 43 and Ret 9, the common primers Ret-NF and Ret-NR were used for the amplification and detection of c-Ret 41 Fig.3.3 Amplification of five log dilutions of standards plasmids pGFRα1a and H2O control 44 Fig 3.4 Agarose gel electrophoresis of amplified products by real time PCR 45 Fig.3.5 Optimization of the specific probe concentration for the sequence dependent real time PCR 46 Fig.3.6 Real time PCR detection using GFRα1a specific MBG probe 47 Fig.3.7 Amplification and detection of GFRα1a and GFRα1b using specific exon overlapping primers 49 Fig.3.8 Amplification of GAPDH and c-Ret standards by real time PCR 50 Fig.3.9 Post PCR analysis 52 Fig.3.10 GFRα1 isoforms and c-Ret expression levels in various human tissues 53 Fig.4.1 Expression levels of GFRα1a, GFRα1b and c-Ret in the transfected cell lines, N-pIRES, N-GFRα1a and N-GFRα1b respectively 61 ix ... effects of GDNF 1. 5 GFR? ?1 9 10 11 1. 5 .1 GFR? ?1 and its spliced isoforms 11 1. 5.2 Expression and functional role of GFRα1a 12 ii 1. 6 Alternatively spliced isoforms 13 1. 7 Quantification of gene expression. .. of The Two GFR? ?1 Spliced Receptor Isoforms 54 4 .1 Introduction 55 4.2 Materials and Methods 56 4.2 .1 Stably transfected cell lines of the two GFR? ?1 spliced isoforms 56 4.2.2 Quantitation of expression. .. Fig.4 .1 Expression levels of GFRα1a, GFRα1b and c-Ret in the transfected cell lines, N-pIRES, N-GFRα1a and N-GFRα1b respectively 61 ix Fig.4.2 Expressions of c-Ret in the three transfected cell lines